Abstract The covalent modification of prokaryotic cell envelope glycans, namely lipopolysaccharide (LPS) and (lipo)teichoic acids, with discrete substituents such as sugars, amino acids, phosphates or acyl groups, is a well-known strategy used by Gram-negative and Gram-positive bacteria to modulate their cell surface properties, the way they interact with their environment, their resistance to biocides and host defenses, and pathogenicity. Although evidence exists that Mycobacterium tuberculosis (Mtb) similarly decorates its major cell envelope glycans, arabinogalactan (AG) and lipoarabinomannan (LAM), with various tailoring substituents, little is known of their biological significance. The discovery by our laboratories of the biosynthetic machineries responsible for the synthesis and transfer of these discrete motifs to AG and LAM, and the generation of the first Mtb knock-out mutants deficient in their synthesis have opened the way to studies aimed at understanding the function of these motifs in the physiology and immunopathogenesis of Mtb. We hypothesize that Mtb has evolved to modify its cell-envelope glycans with a distinct array of strategically placed substituents to promote its survival in the host environment. Accordingly, a multidisciplinary team of investigators with complementary expertise in mycobacterial cell envelope genetics and glycobiology, TB immunopathogenesis, and carbohydrate chemistry here proposes to investigate how simple (amino/thio)sugars or other charged groups strategically placed within the Mtb cell envelope landscape affect not only the physiology of this microorganism (Aim 1), but also its pathogenicity in different in vivo models of TB infection (Aim 2) and its interactions with host macrophages and dendritic cells thereby promoting survival within the host (Aim 3). Ultimately, these studies are expected to lead to significant new knowledge about the biological significance of understudied aspects of the unique cell wall of mycobacteria.